Reagents for automated synthesis of peptide analogs

ABSTRACT

Reagents suitable for synthesis of peptide analogs using automated peptide synthesis and procedures for synthesis of peptide analogs are provided.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation-in-part of U.S. Ser. No. 07/627,753,filed Dec. 14, 1990, now U.S. Pat. No. 5,283,293the disclosure of whichis incorporated herein by reference.

FIELD OF THE INVENTION

This invention relates to methods for the automated solid-phasesynthesis of peptide analogs and to novel reagents useful therein. Inone aspect, the present invention is directed to a process which usesnovel reagents which comprise novel heterobifunctional semicarbazide(I), or semicarbazone (II) or (III) linker moieties which may beattached to insoluble resins (or supports), via a pendant carboxylicacid group to give a support reagent suitable for automated solid phasesynthesis of peptide analogs. The resulting support reagent is suitablefor use in a conventional automated or semi-automated peptidesynthesizer using protected amino acids or amino acid analogs, to give aprotected peptide (or peptide analog) aldehyde, attached to the supportreagent. The product peptide aldehyde or peptide analog is cleaved fromthe support and deprotected to give the desired peptide analog in goodyield. Using this process and reagents of the present invention, peptidealdehydes and analogs can be rapidly and efficiently produced. Thesepeptide analogs are useful as enzyme inhibitors and have potential aspharmaceutical agents.

BACKGROUND OF THE INVENTION

Analogs which utilize the catalytic mechanism of an enzyme (e.g.transition-state inhibitors) have been suggested as enzyme inhibitors,however it has only been recently that this idea has been explored. Amajor problem has been the difficulty in synthesizing the target peptideanalog molecules. One candidate group are analogs having the aldehydegroup. These peptide analogs are particularly attractive in that theycan be prepared from naturally occurring amino acids. Highly specificand potent peptide transition-state analog enzyme inhibitors would be ofinterest as therapeutic agents. Solution methods for the synthesis ofpeptide aldehydes have been developed, however, their preparationremains a tedious, labor intensive, and time consuming process. Thedevelopment of a methodology for the automated synthesis of thesealdehyde derivatives will allow for the rapid synthesis of a largenumber of these analogs and, thus, would facilitate the exploration ofenzyme inhibition structure-activity relationships. The availability ofsuch analogs would facilitate the development of drugs that selectivelyinhibit specific serine or cysteine proteinases.

The serine proteinases may be suitable targets for inhibition by peptidetransition-state analogs. The trypsin sub-family is composed of serineproteinases which hydrolyse peptide bonds that follow an arginine orlysine residue. Trypsin-like enzymes play a physiological role indigestion, coagulation, fibrinolysis, blood pressure regulation,fertility, and inflammation (see: "Design of Enzyme Inhibitors as Drugs"Eds. Sandler, M., Smith, H. J., Oxford Science Publications, 1989).Selective inhibitors of members of this family of enzymes may thereforebe useful in the intervention of many disease states. The catalyticmechanism of serine proteinases involves the attack of the active-siteserine on the carbonyl bearing the sissile amide bond of the substrate,to give a tetrahedral intermediate. It has been reported that peptideanalogs which are stable mimics of this tetrahedral intermediate (i.e.,transition-state analogs) can be selective enzyme inhibitors (seeDelbaere, L. T. J., Brayer, G. D., J. Mol. Biol. 183:89-103, 1985 andAoyagi, T., Umezawa, H., Eds., Proteases and Biological Control, ColdSpring Harbor Laboratory Press, 429-454, 1975). Methods for identifyingpotent and selective inhibitors (as potential drugs) is an active areaof research.

Peptide aldehydes were initially discovered as natural products producedby a number of actinomycete strains. Some of these derivatives have beereported to be selective inhibitors of various types of serine andcysteine proteinases (see Aoyagi, T. et al., cited above). For example,the peptide alaninal elastatinal is a potent elastase inhibitor, whilenot inhibiting trypsin or trypsin-like enzymes (see Hassall, C. H. etal., FEBS Lett., 183:201-5, 1985). In several cases, the selectivity ofthese naturally occurring analogs has been enhanced by modifying thesequence. (See, e.g., Bajusz, S. et al., J. Med. Chem. 33:1729-1735,1990, and McConnell, R. M. et al., J. Med. Chem. 33:86-93, 1990.)Elastase inhibitors are of interest in the treatment of diseases such anemphysema and synthetic peptide aldehydes have been reported to beexcellent inhibitors of human leucocyte elastase (see "Design of EnzymeInhibitors as Drugs" cited above). The peptide arginal leupeptin hasbeen reported to be a selective inhibitor of trypsin-like enzymes (seeAoyagi, T., Umezawa, H., Eds., "Structures and activities of proteaseinhibitors of microbial origin", Proteases and Biological Control, ColdSpring Harbor Laboratory Press, 429- 454, 1975). Leupeptin, along withnaturally occurring variants and synthetic analogs, has been reported tobe potent inhibitors of several trypsin like enzymes in the coagulationcascade. Synthetic peptide analogs have been prepared which are reportedto show a marked selectivity for particular coagulation factors. Forexample, one such analog (Me-D-Phe-Pro-Arg-al) has been developed as athrombin inhibitor and is reported to have significant in vivoanticoagulant activity. (See U.S. Pat. Nos. 4,316,889 (1982), 4,399,065(1983), 4,478,745 (1984), 4,346,078 (1982), and 4,708,039 (1987).)

Resins for the affinity isolation of specific enzymes have been reportedwhich have unprotected peptide and amino acid aldehydes attached toinsoluble supports. (See Patel et al., Biochem, Biophys. Res. Comm. 104,181-186 (1982) and Patel et al., Biochem. Biophys. Acta, 748, 321-330(1983).) Those resins were neither intended nor suitable for use in thesolid phase synthesis of peptide aldehydes, since the support wasattached to the N-terminus of the peptide aldehyde.

Methods for the solution synthesis of peptide aldehydes have beenreported. See, e.g., McConnell et al. and references therein; andBajusz, S. et al. both cited above; Kawamura et al., Chem, Pharm. Bull.,17: 1902 (1969), and Someno et al., Chem. Pharm. Bull. 34, 1748, (1986).The use of semicarbazides as aldehyde protecting reagents for thesolution synthesis of peptide aldehydes has also been reported. Westerikand Wolfnden, J. Biol. Chem., 247, 8195 (1972), Ito et al., Chem. Pharm.Bull. 23, 3081, (1975), and McConnell et al. (cited above). The use of asoluble semicarbazide functionalized polymer has been reported for themanual preparation of some peptide aldehydes. Galpin et al., (Pept.Struct. Funct. Proc. Am Pept. Symp., 9th, 799-802 (1985). Edited by:Deber, C. M., Hruby, V. J., Kopple, K. D., Pierce Chem. Co.: Rockford,Ill.). However, such supports were not suitable for the automaticsynthesis of peptide aldehydes, since they dissolve in the solvents usedfor the coupling steps.

SUMMARY OF THE INVENTION

The present invention relates to methods for the automated synthesis ofpeptide analogs and for reagents useful for such methods. Such synthesescan be performed on conventional automated peptide synthesizers, usingthe novel solid insoluble support reagents (hereafter referred to as"semicarbazone (or semicarbazide) amino acid aldehyde supports" or "SAAAsupports") of the present invention. The present invention also relatesto novel linker moieties used to prepare the SAAA supports. These linkermoieties have the general structure: ##STR1## wherein (a) A is adivalent spacer group which comprises a non-reactive divalenthydrocarbyl group having from 2 to about 15 carbon atoms; and

(b) Z is ##STR2## wherein Pr is a protecting group removable undernon-adverse conditions; R₁ is hydrogen, or alkyl of 1 to 12 carbonatoms, cycloalkyl of 5 to 8 carbon atoms, aryl or aralkyl of about 7 toabout 15 carbon atoms, all optionally substituted with 1 to 3 groupsindependently selected from hydroxy, sulfhydryl, alkylthio, carboxyl,amide, amine, alkylamine, idolyl, 3-N-formylindolyl, benzyloxy,halobenzyloxy, guanido, nitroguanido or imidazolyl optionallysubstituted with alkoxyalkyl; alk is an alkylene group of about 3 toabout 12 carbon atoms optionally substituted with 1 to 3 substituentsindependently selected from hydroxy, alkyl, aryl or guanido and providedthat any functional groups of R₁ or alk which are reactive underconditions of peptide synthesis are optionally protected by a protectinggroup which is removable under non-adverse conditions.

Suitable protecting groups, Pr, include t-butoxycarbonyl (BOC),9-fluorenylmethyloxycarbonyl (FMOC) and other suitable protectinggroups.

Suitable non-reactive hydrocarbyl groups, A, are those which aresubstantially inert (and may have suitably protected functional groups)under conditions for automated and semi-automated peptide synthesis.Preferably A has from about 5 to about 10 carbon atoms. A is preferablya divalent C₅ -C₈ cycloalkylene group optionally substituted with 1 toabout 5 alkyl groups such as a 1,4-cyclohexylene group, or a divalent C₅-C₈ arylene or aralkylene group optionally substituted with 1 to about 5alkyl groups such as a 1,3- or 1,4-phenylene radical, an alkyl-1,3- or1,4-phenylene radical or the like.

Preferably R₁ is hydrogen, or a protected or unprotected amino acid sidechain. Particularly suitable amino acid side chains include side chainsselected from those of the following amino acids: glycine, alanine,valine, cysteine, leucine, isoleucine, serine, threonine, methionine,glutamic acid, aspartic acid, glutamine, asparagine, lysine, arginine,histidine, phenylalanine, tyrosine, and tryptophan. Preferred alk groupsinclude propylene to give the side chain of the amino acid proline.Where the amino acid side chain contains functional groups which arereactive under conditions for peptide synthesis, those groups arepreferably protected by suitable protecting groups which are removableunder non-adverse conditions. Other suitable R₁ or alk groups includethe side chains of the following amino acids: hydroxyproline,norleucine, 3-phosphoserine, homoserine, O-phosphohomoserine,dihydroxyphenylalanine, 5-hydroxytryptophan, 1-methylhistidine,3-methylhistidine, and β-aspartyl phosphate. Other naturally occurringamino acid metabolites or precursors having side chains which aresuitable for use as R₁ groups herein include α-amino-adipic acid,cysteine sulfonic acid, cysteic acid and ornithine. As noted above,preferred alk groups include propylene, optionally substituted withhydroxy to give a proline or hydroxyproline analog.

In the SAAA supports (or "support reagents") of the present invention,the carbon of the C-terminal carboxyl group is attached to a solidsupport or resin or such that the hydroxy group of the C-terminalcarboxyl group is replaced by --X wherein --X is independently selectedfrom --NH--sp, --O--Sp, and --CH₂ --Sp, wherein Sp denotes an insolublesupport, preferably a suitably functionalized 1% cross-linkedpolystyrene.

The support reagents of the present invention are conveniently preparedusing protecting groups suitable for chemically extending the peptidechain by conventional automated solid phase techniques. Such protectinggroups include, but are not limited to t-butoxycarbonyl (BOC) and9-fluorenylmethyloxycarbonyl (FMOC), allyloxycarbonyl and the like. Seefor example, Green, T.; "Protecting Groups in Organic Synthesis," (JohnWiley and Sons, 1981). For a general description of peptide synthesis,see Greenstein, J. P., Winitz, M. "Chemistry of the Amino Acids," pages763-1268. (John Wiley and Sons: New York, 1986).

Once the peptide transition state analog has been extended on the SAAAsupport to give the desired sequence and chain length, the analog can becleaved from the support by mild acid/formaldehyde treatment. Theprotecting groups on the N-terminus and side-chains of the amino acidcomponents of the analog are selected so that they are not affected bycleavage of the peptide analog from the support. If desired, acid orbase sensitive protecting groups can be removed before cleavage. Adeprotection step involving catalytic hydrogenation after cleavage ofthe peptide analog from the support may be used, since many protectinggroups (e.g., benzyl, benzyloxycarbonyl, benzyloxymethyl,halobenzyloxycarbonyl, and nitor) are readily removed by mildhydrogenolysis without affecting functionality of the desired finalproduct (see, e.g., Example 8).

In another aspect, the present invention is directed to peptide analogsof the formula: ##STR3## wherein Pr is a protecting group removableunder non-adverse conditions, Res is an independently selected aminoacid residue, n is an integer greater than zero, W is ##STR4## whereinR₁ is hydrogen; or alkyl, cycloalkyl, aryl or aralkyl, optionallysubstituted with 1 to 3 substituents independently selected fromhydroxy, alkoxy, sulfhydryl, alkythio, carboxyl, amide, amino,alkylamino, indolyl, 3-N-forylindolyl, benzyloxy, halobenzyloxy,guanido, nitro-guanido- or optionally substituted imidazolyl substitutedwith alkoxy-alkyl; and alk is an alkylene group of about 3 to about 12carbon atoms optionally substituted with 1 to 3 substituentsindependently selected from hydroxy, alkyl, aryl or guanido; A is anon-reactive hydrocarbyl group of 2 to about 15 carbon atoms; and X isindependently --NH--Sp, O--Sp, or --CH₂ --Sp, where Sp is an insolubleresin support; and provided that any functional groups of Res, R₁ or alkwhich are reactive under conditions of peptide synthesis are optionallyprotected by a protecting group which is removable under non-adverseconditions, which are conveniently prepared using the linker moietiesand support reagents of the present invention.

Preferred A groups are those having 5 to 10 carbon atoms. Morepreferably, A is a divalent C₅ -C₈ cycloalkylene group, a C₅ -C₈divalent arylene group or a C₅ -C₈ divalent aralkylene group, alloptionally substituted with 1 to 5 alkyl groups. Especially preferred Agroups include 1,3-cyclohexylene, 1,4-cyclohexylene, 1,3-phenylene,1,4-phenylene, 1,3-xylylene, 1,4-xylylene and the like.

Preferred R₁ and alk groups are those which correspond to the sidechains of the amino acids typically found in proteins, i.e., glycine,alanine, valine, leucine, isoleucine, serine, threonine, cysteine,methionine, glutamic acid, aspartic acid, glutamate, aspartate, lysine,arginine, histidine, phenylalanine, tyrosine, tryptophan and proline.Also preferred are amino acid residues, Res, having R₁ or alk groupswhich comprise those amino acid side chains.

Preferred are those compounds wherein n is less than 10 (to give adecamer after cleavage) or more preferably less than 5 (to give apentamer).

An additional aspect of the present invention is directed to methods ofpreparing the above peptide analog (V) using the linker moieties andsupport moieties of the present invention. After cleavage anddeprotection, a peptide aldehyde of the formula

    H--(--Res--).sub.n --W--CHO                                (VI)

is obtained, wherein Res and W are as defined in conjunction withformula V after removal of any protecting groups. These methods areparticularly suitable for preparing peptide analogs where n is less than10 (a decamer) or less than 5 (a pentamer).

Definitions

As used herein, the following terms have the following meanings, unlessexpressly stated to the contrary:

The nomenclature used to define the peptides is that specified bySchroder & Lubke, "The Peptides," Academic Press (1965), wherein inaccordance with conventional representation the amino group at theN-terminus appears to the left and the carboxyl group at the C-terminusto the right.

The term "amino acid residue" refers to radicals having the structure(i) --C(O)RNH-- wherein R typically is --CH(R₁)-- and R₁ is H or acarbon containing substituent, or (ii) ##STR5## wherein alk is analkylene group. For the most part, the amino acids used in theapplication of this invention are those naturally occurring amino acidsfound in proteins,or the naturally occurring anabolic or catabolicproducts of such amino acids which contain amino and carboxyl groups.Also included are the D and L stereoisomers of such amino acids when thestructure of the amino acid admits of stereoisomeric forms. For thepurposes of this application, unless expressly noted to the contrary, anamed amino acid shall be construed to include both the D or Lstereoisomers, preferably the L stereoisomer.

The term "hydrocarbyl" denotes an organic radical composed of carbon andhydrogen which may be aliphatic (including alkyl, alkenyl, and alkynylgroups and groups which have a mixture of saturated and unsaturatedbonds), alicyclic (carbocyclic), aryl (aromatic) or combinationsthereof; and may refer to straight-chained, branched-chain, or cyclicstructures or to radicals having a combination thereof, as well as toradicals substituted with halogen atom(s) or heteroatoms, such asnitrogen, oxygen and sulfur and their functional groups (such as amino,alkoxy, aryloxy, carboxyl, ester, amide, carbamate or lactone groups,and the like), which are commonly found in organic compounds andradicals.

The term "hydrocarbylcarbonyl" refers to the group ##STR6## wherein R'is a hydrocarbyl group.

The term "alkyl" refers to saturated aliphatic groups, includingstraight, branched and carbocyclic groups.

The term "aryl" refers to aromatic hydrocarbyl and heteoaromatic groupswhich have at least one aromatic ring.

The term "aralkyl" refers to an alkyl group which has been substitutedwith an aromatic (or aryl) group, and includes, for example, groups suchas benzyl.

The term "alkylene" refers to straight and branched-chain alkylenegroups which are biradicals, and includes, for example, groups such asethylene, propylene, 2-methylpropylene (e.g. ##STR7## and the like.

The term "arylene" refers to aromatic groups which are biradicals.

The term "aralkylene" refers to aralykyl groups which are biradicals.

The term "ester" refers to a group having a ##STR8## linkage, andincludes both acyl ester groups and carbonate ester groups.

The term "halo" or "halogen" refers to fluorine, chlorine, bromine andiodine.

The term "non-adverse conditions" describes conditions of reaction orsynthesis which do not substantially adversely affect the skeleton ofthe peptide analog and/or its amino acid (and/or amino acid analog)components. One skilled in the art can readily identify functionalities,coupling procedures, deprotection procedures and cleavage conditionswhich meet these criteria.

The term "support" refers to a solid particulate, insoluble material towhich a linker moiety of the present invention is linked and from whicha peptide analog may be synthesized. Supports used in synthesizingpeptide analogs are typically substantially inert and nonreactive withthe reagents used in the synthesis of peptide analogs and include resinssuch as that included in the SAAA support reagents of the presentinvention.

The term "peptide analog" refers oligomers of amino acids (or amino acidresidues) which are linked by peptide linkages wherein either theC-terminal carboxyl or the N-terminal amino has been chemically modifiedto another functional group or replaced with a different functionalgroup. For example the C-terminal carboxyl group may be replaced with analdehyde group.

The term "automated synthesis" or "automated peptide synthesis" refersto the synthesis of peptides or peptide analogs using an instrumentwhich carries out the individual steps of each addition cycle to add aamino acid or amino acid analog to the growing peptide chain occurswithout manual manipulation.

The term "semi-automated synthesis" or semi-automated peptide synthesisrefers to the synthesis of peptides or peptide analogs where in eachaddition cycle to add an amino acid or amino acid analog to a growingpeptide chain, the coupling step is done manually and other individualsteps occur without manual manipulation. Thus, semi-automated synthesisdiffers from automated synthesis in that some of the coupling steps arecarried out manually.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 depicts general reaction schemes for the preparation of certainsome reagents according to the present invention.

FIGS. 2A and 2B depict reaction schemes for the preparation of supportreagents of the present invention.

FIG. 3 depicts a reaction scheme for the preparation of α-protectedamino acid analogs which may be used in the preparation of the supportreagents of the present invention.

DETAILED DESCRIPTION OF THE INVENTION

Accordingly, aspects of the present invention are directed to processesfor the synthesis of support reagents which comprise novel linkermoieties and the peptide analogs derived therefrom, as well as processeswhereby transition-state peptide analogs are prepared using said supportreagents.

A. Peptide Synthesis

Techniques for solid-phase peptide synthesis are described in"Solid-Phase Peptide Synthesis," Steward & Young, (Freeman & Co., SanFrancisco, 1969) and U.S. Pat. No. 4,105,603, issued Aug. 8, 1978.Classical solution synthesis methods are described in detail in thetreatise "Methoden der Organischen Chemie-(Houben-Weyl) Synthese vonPeptiden," E. Wunsch (ed.) (1974), (Georg Thieme Verlag, Stuttgart, W.Ger). The fragment condensation method of synthesis is described in U.S.Pat. No. 3,972,859 issued Aug. 3, 1976; other synthesis methods aredescribed by U.S. Pat. No. 3,842,067 issued Oct. 15, 1974 and U.S. Pat.No. 3,862,925 issued Jan. 28, 1975, the disclosures of which areincorporated herein by reference.

The peptide chain of the peptide analog to be synthesized may beextended using solid-phase synthesis methods, such as that generallydescribed by Merrifield, J. Am. Chem. Soc. 85:2149 (1963); however otherchemical syntheses protocols known in the art may be used. Solid-basesynthesis is initiated from the C-terminus of the peptide aldehyde oranalog to be synthesized by coupling a protected α-amino acid to asuitable SAAA support. Examples of supports or resins which are suitablefor the preparation of SAAA supports of the present invention includeMBHA, BHA, aminomethyl phenyl resins and the like. The preparation ofthe hydroxymethyl resin is described by Bodansky et al., Chem. Ind.38:1597-1598 (London) 1966. Chloromethylated resins are commerciallyavailable from BioRad Laboratories, Richmond, Calif. and from Lab.Systems, Inc. The preparation of resins is described by Stewart et al.,"Solid Phase Peptide Synthesis," Chapter 1, pages 1-6 (Freeman & Co.,San Francisco 1969). BHA and MBHA resin supports are commerciallyavailable, but have conventionally been used only when the desiredpolypeptide being synthesized has an unsubstituted amide at theC-terminus.

The peptide chain is extended by coupling additional amino acids to thechain using known techniques for the formation of peptide bonds. Onesuitable method comprises converting the α-amino protected amino acid tobe added to the peptide chain to an "activated" derivative wherein thecarboxyl group is rendered more susceptible to reaction with the freeN-terminal α-amino group of the peptide fragment. For example, the aminoacid can be converted to a mixed anhydride by reaction of a protectedamino acid with ethyl chloroformate, pivaloyl chloride or like acidchlorides. Alternatively, the amino acid can be converted to an activeester such as a 2,4,5-trichloropheyl ester, a pentachlorophenol ester, apentafluorophenyl ester, a p-nitrophenyl ester, a N-hydroxysuccinimideester, or an ester formed from 1-hydroxybenzotriazole.

Another coupling method involves use of a suitable coupling agent suchas N,N'-dicyclohexylcarbodiimide or N,N'diisopropylcarbodiimide. Otherappropriate coupling agents are disclosed in E. Gross & J. Meinenhofer,The Peptides: Analysis, Structure, Biology, Vol. I: Major Methods ofPeptide Bond Formation (Academic Press, New York, 1979).

The α-amino groups of amino acids (monomers) employed in the peptidesynthesis are protected during the coupling reaction to prevent sidereactions involving the reactive, if unprotected, α-amino function. Inaddition, certain amino acids contain reactive side-chain functionalgroups (e.g., sulfhydryl, amino, carboxyl, and hydroxyl) which must alsobe protected with suitable protecting groups to prevent chemicalreaction of those groups from occurring during both the initial andsubsequent coupling steps. Suitable protecting groups, known in the art,are described in E. Gross & J. Meienhofer, The Peptides: Analysis,Structure, Biology, Vol. 3: Protection of Functional Groups in PeptideSynthesis (Academic Press, New York, 1981).

In selecting a particular side-chain protecting group to be used duringsynthesis of the peptide analogs, the following considerations may bedeterminative. An α-amino protecting group (a) should render the α-aminofunction inert under the conditions employed in the coupling reaction,(b) should be readily removable after the coupling reaction underconditions that will not remove side-chain protecting groups and willnot alter the structure of the peptide fragment, and (c) shouldeliminate the possibility of racemization upon activation immediatelyprior to coupling. An amino acid side-chain protecting group (a) shouldrender the side chain functional group inert under the conditionsemployed in the coupling reaction, (b) should be stable under theconditions employed in removing the α-amino protecting group, and (c)should be readily removable upon completion of the desired amino acidpeptide aldehyde under reaction conditions that will not alter thestructure of the peptide analog chain.

It will be apparent to those skilled in the art that the protectinggroups known to be useful for peptide synthesis will vary in reactivitywith the agents employed for their removal. For example, certainprotecting groups such as triphenylmethyl and2-(p-biphenylyl)isopropyloxycarbonyl are very labile and can be cleavedunder mild acid conditions. Other protecting groups, such ast-butyloxycarbonyl (BOC), t-amyloxycarbonyl, adamantyloxycarbonyl, andp-methoxybenzyloxycarbonyl are less labile and require moderately strongacids, such as trifluoroacetic, hydrochloric, or boron trifluoride inacetic acid, for their removal. Still other protecting groups, such asbenzyloxycarbonyl (CBZ), halobenzyloxycarbonyl, p-nitrobenzyloxycarbonylcycloalkyloxycarbonyl, and isopropyloxycarbonyl, are even less labileand require stronger acids, such as hydrogen fluoride, hydrogen bromide,or boron trifluoroacetate in trifluoroacetic acid, for their removal.

Example of amino acid protecting groups include:

(1) for an α-amino group, (a) aromatic urethane-type protecting groups,such as fluorenylmethyloxycarbonyl (FMOC); (b) aliphatic urethane-typeprotecting groups, such as BOC, t-amyloxycarbonyl, isopropyloxycarbonyl,2-(p-biphenylyl)isopropyloxycarbonyl, allyloxycarbonyl and the like; (c)cycloalkyl urethane-type protecting groups, such ascyclopentyloxycarbonyl, adamantyloxycarbonyl, and cyclohexyloxycarbonyl;and (d) allyloxycarbonyl. The preferred α-amino protecting groups areBOC or FMOC.

(2) for the side chain amino group present in Lys, protectinggroups--include any of the groups mentioned above in (1) such as BOC,p-chlorobenzyloxycarbonyl, etc.

(3) for the guanidino group of Arg, protecting groups preferably includenitro, or 2,2,5,7,8-pentamethylchroman-6-sulfonyl or2,3,6-trimethyl-4-methoxyphenylsulfonyl.

(4) for the hydroxyl group of Ser, Thr, or Tyr, protecting include, forexample, t-butyl; benzyl (BZL); substituted BZL groups, such asp-methoxybenzyl, p-nitrobenzyl, p-chlorobenzyl, o-chlorobenzyl, and2,6-dichlorobenzyl.

(5) for the carboxyl group of Asp or Glu, protecting groups include, forexample, by esterification using groups such as t-butyl, or preferablybenzyl.

(6) for the imidazole nitrogen of His, suitable protecting groupsinclude the benzyloxymethyl group

(7) for the phenolic hydroxyl group of Tyr, protecting groups such astetrahydropyranyl, tert-butyl, trityl, benzyl, chlorobenzyl,4-bromobenzyl, and 2,6-dichlorobenzyl are suitably employed. Thepreferred protecting group is bromobenzyloxycarbonyl.

(8) for the side chain sulfhydryl group of cysteine, trityl ispreferably employed as a protecting group.

Following the coupling of the BOC-protected amino acid to the supportreagent,the α-amino protecting group is removed, such as by usingtrifluoroacetic acid (TFA) in methylene chloride or TFA alone. Thedeprotection is carried out at a temperature of from about 0° C. toabout ambient temperature. Other suitable cleaving reagents, such as HClin dioxane, and conditions for removal of specific α-amino protectinggroups are described in Shroder & Lubke, supra, Chapter I, pages 72-75.

After the α-amino protecting group is removed, the remaining α-amino andside-chain protected amino acids are coupled stepwise in the desiredorder. As an alternative to adding each amino acid separately in thesynthesis, some may be coupled to one another prior to their addition tothe solid-base synthesizer so as to give a dipeptide or tripeptideanalog. The selection of an appropriate coupling reagent is within theskill of the art. Particularly suitable as a coupling reagent isN,N'-dicyclohexyl carbodiimide or diisopropylcarbodiimide.

Each protected amino acid or amino acid sequence to be coupled to thegrowing peptide sequence or chain is introduced into the solid-phasereactor in excess, and the coupling is suitably carried out in a mediumof suitable solvent such as dimethylformamide (DMF) or CH₂ Cl₂ ormixtures thereof. If incomplete coupling occurs, the coupling procedureis repeated before removal of the α-amino protecting group of theproduct peptide sequence prior to the coupling of the next amino acid.The success of the coupling reaction at each stage of the synthesis maybe monitored. A preferred method of monitoring the synthesis uses by theninhydrin reaction, as described by Kaiser et al., Anal. Biochem 34:595(1970). The coupling reactions can be performed automatically using wellknown methods and instruments, for example, a Biosearch 9550 PeptideSynthesizer or Applied Biosystems Model 430A peptide synthesizer.

Upon completion of the desired peptide sequence, the protected peptidealdehyde is cleaved from the resin support, and protecting groups areremoved. The cleavage reaction and removal of the protecting groups maybe suitably accomplished simultaneously or stepwise. It will also berecognized that the protecting groups present on the N-terminal α-aminogroups may be removed preferentially either before or after theprotected peptide is cleaved from the support.

Purification of the polypeptide analogs prepared using the reagents ofthe present invention is typically achieved using conventionalprocedures such as preparative HPLC (including reversed phase HPLC) orother known chromatography, affinity chromatography (includingmonoclonal antibody columns) or countercurrent distribution.

B. General Preparation of Support Reagents (FIG. 2)

FIG. 2 depicts alternative generalized reaction schemes for thepreparation of support reagents of the present invention. Blockinggroup, B1, denotes a protecting group which is removable undernon-adverse conditions.

According to FIG. 2A, protected semicarbazide moiety (21) is reactedwith support (22) to give protected intermediate (23). The protectinggroup of intermediate (23) is removed under nonadverse conditions togive deprotected intermediate (24). Deprotected intermediate (24) isthen reacted with α-aminoprotected amino acid aldehyde (25) or (25a) togive the appropriate support regent (26) or (26a) in a reaction whichpreserves the chirality of the amino acid aldehyde. Although FIG. 2Adoes not depict the stereochemical configuration of amino acid aldehyde(25), if either the L-isomer the D-isomer (as opposed to a racemicmixture) is employed, the stereochemistry of its chiral center will beconserved.

According to FIG. 2B, the protecting group, Pr, of protectedsemicarbazide moiety (21) is removed under non-adverse conditions.Deprotected semicarbazide moiety (27) is reacted with α-amino protectedamino acid aldehyde (25) or (25a) to give semicarbazone intermediate(28) or (28a), as appropriate. The resulting intermediate is reactedwith support (22) to give the appropriate support regent (26) or (26a)in a reaction which maintains the stereochemistry of the chiral centerof amino acid aldehyde (25). Thus, if either the D- or the L- isomer of(25) (as opposed to a racemic mixture) is used, the stereochemistry ofthe chiral center will be conserved.

C. Preparation of α-Aminoprotected Amino Acid Aldehydes

The α-aminoprotected amino acid aldehydes (25) and (25a) of FIG. 2 maybe prepared as described in Examples 1 to 18. FIG. 3 depicts onepreferred reaction scheme for preparing aldehydes of formula (25) and(25a). In FIG. 3, blocking group, B1, denotes a protecting group whichis removable under non-adverse conditions.

According to FIG. 3, a mixture of α-amino protected amino acid (31) or(31a), where B1 is a protecting group removable under nonadverseconditions, and methylchloroformate (32) in solvent is reacted with amixture of dimethylhydroxylamine (33) to give the carboxamide (34) or(34c). The carboxamide (34) or (34a) is reacted with lithium aluminiumhydride and then worked-up under acid conditions (36) to given theresulting α-amino protected amino acid aldehyde (25) or (25b).

DESCRIPTION OF PREFERRED EMBODIMENTS

A. Preparation of Preferred Support Reagents Comprising Semicarbizideand Semicarbazone Linker Moieties

FIG. 1 depicts a general reaction scheme for the synthesis of certainpreferred semicarbazide and semicarbazone linker moieties and thesupport reagents incorporating those moieties. The reaction oft-butylcarbazate with carbonyldiimidazole gives an intermediate (havingpresumed structure 3) which is then allowed to react directly withepsilon amino ester (2). Epsilon amino ester (2) may be convenientlyprepared from the commercially available trans-4-aminomethylcyclohexanecarboxylic acid. The resulting protected semicarbazide (4) can beisolated by chromatography or alternatively may be converted directly tocrystalline carboxylic acid (5), without further isolation in anapproximately 62% overall yield. The free semicarbazide salt is preparedby allowing trifluoroacetic acid (TFA) to react with (5).

According to procedure A, carboxylic acid (5) is allowed to react withan insoluble amine resin such as methylbenzhydrylamine (MBHA). Thereaction product is treated successively with TFA and then with theprotected amino acid aldehyde α-t-butoxycarbonyl-N^(g) -nitroarginal (1)to give support (8). Alternatively, other suitably protected amino acidaldehydes may be substituted for (1).

Procedure B depicts an alternative protocol for the preparation ofsupport regents of the present invention. According to Procedure B,preformed semicarbazone (7) is allowed to react directly with the resin(for example an amine resin such as MBHA). Procedure B may be preferredsince support reagents having high substitution of semicarbazone linkingmoiety are readily obtained.

In the preparation of the preferred SAAA support reagents of the presentinvention by attachment to the novel linker moieties, polymeric resinshaving one or more of the following characteristics are particularlysuitable:

(1) Resins which are insoluble in polar aprotic solvents such asdimethyl formamide (DMF), N-methylpyrrolidone, tetrahydrofuran (THF) andother solvents conventionally used in solid-phase peptide synthesis(such as dichloromethane or methanol).

(2) Resins which are microporous and which have high surface areas inpolar aprotic solvents.

(3) Resins which are capable of being functionalized with groups whichcan react with a carboxylic acid group to form a bond which is stableduring the subsequent addition of amino acids (or amino acid analogs) tothe N-terminal end of the growing peptide chain.

(4) Resins which are stable in the presence of reagents such as TFA,diisopropylethyl amine, dicyclohexylcarbodiimide (DCC) and otherreagents conventionally used in solid phase peptide synthesis.

Suitable resins having the above properties include commerciallyavailable resins which include P-methyl-(benzhydrylamine) (MBHA) oraminomethylated 1% divinylbenzene crosslinked polystyrene resins. Othersuitable functionalized supports include pellicular and macroporousinsoluble supports known to those skilled in the art (See, e.g., G.Barany and R. B. Merrifield, "Solid-Phase Peptide Synthesis," in ThePeptides, Volume 2, pages (1-2984 (Academic Press, New York 1980)).

B. Preparation of Peptide Analogs Using Preferred Support Reagents

In the preparation of peptide aldehydes, SAAA resin (8) is added to thereaction vessel of an automated or semi-automated synthesizer. In thefirst synthesis, the t-butoxycarbonyl (BOC) protecting group on anα-amino group is removed with an acid such as TFA to give the resultingfree amine. The amino group is coupled to a suitably activated carboxylgroup of a blocked amino acid, using a suitable reagent such as DCC. Theresulting resin undergoes a series of washing steps between eachreaction. The addition cycle can then be repeated using the nextα-blocked amino acid until the desired peptide sequence is complete.When the sequence is complete, the peptide aldehyde or analog may bereleased from the support (resin) by cleavage methods such as bytreatment with aqueous formaldehyde and dilute acid to give theprotected peptide aldehyde (or analog). If the product peptide analoghas side chain or N-terminal protecting or blocking groups, these groupsmay be removed by conventional procedures such as by treatment withhydrogen and palladium catalysts (See Example 8). The peptide aldehydesmay be further purified using procedures such as HPLC (See Examples 8 etseq.).

To assist in understanding the present invention, the following examplesfollow, which include the results of a series of experiments Thefollowing examples relating to this invention are illustrative an shouldnot, of course, be construed as specifically limiting the invention.Moreover, such variations of the invention, now known or laterdeveloped, which would be within the purview of one skilled in the artare to be considered to fall within the scope of the present inventionhereinafter claimed.

EXAMPLES EXAMPLE 1 Preparation of α-N-t-butoxycarbonyl-N^(g)-nitroargininal ##STR9##

The following procedure for the synthesis ofalpha-t-butoxycarbonyl-N^(g) -nitro-argininal is an example of a generalprocedure for the preparation of BOC-amino acid aldehydes, See Patel etal., Biochim. Biophys. Acta, 748, 321-330 (1983).

In 200 ML dry THF, 12.7 g BOC-N^(g) -nitro-arginine (40 mmoles) and 7.0g carbonyldiimidazole (CDI, 43 mmoles) were added at room temperatureand allowed to stir for 30 minutes The reaction mixture was cooled to-78° C. and 35 mmoles of LiAlH₄ (1M in THF) were added dropwise overthirty minutes. The reaction mixture was allowed to stir for anadditional hour at -78° C. Next, 18 mL of acetone was added and theresulting mixture was quickly added to 400 mL of 1N HCl. The mixture wasextracted twice with 100 mL of ethyl acetate. The ethyl acetate washeswere combined and then washed two times each with 100 mL water, 100 mLsaturated NaHCO₃ and 100 mL saturated NaCl (brine). The solution wasdried (MgSO₄) and concentrated to a foam. The crude weight of thealpha-t-butoxycarbonyl-N^(g) -nitro-argininal was 6.36 g (21 mmole;yield 52%).

EXAMPLE 1A Preparation of α-N-t-butoxycarbonyl-N^(g) -nitroargininal##STR10##

The following procedure for the synthesis ofalpha-t-butoxycarbonyl-N^(g) -nitro-argininal (1) is a modification ofthe procedure of Fehrentz, J. A. and Castro, B., Synthesis, 676 (1983).

BOC-N^(g) -nitroarginine was obtained from Calbiochem.N-methylpiperidine, N,O-dimethylhydroxylamine hydrochloride andisobutylchloroformate, and lithium aluminum hydride were obtained fromAldrich chemical Company, Inc. Dichloromethane, ethyl acetate, methanoland tetrahydrofuran were obtained from Fisher Scientific Company.

11.4 mL of N-methylpiperidine was slowly added to a stirred suspensionof 8.42 g (94 mmole) of N,O-dimethylhydroxylamine in 75 mLdichloromethane which had been cooled to about 0° C. The solution wasallowed to stir for 20 minutes which gave the free hydroxylamine, thenwas kept cold for use in the next step.

In a separate flask, 30.0 g (94 mmole) of Boc-N^(g) -nitroarginine wasdissolved by heating in about 1400 mL of tetrahydrofuran and cooledunder nitrogen to 0° C. 11.4 mL of N-methylpiperidine and 12.14 mL (94mmole) of isobutylchloroformate was added and the mixture stirred for 10minutes. The free hydroxylamine prepared above was added all at once andthe reaction mixture was allowed to warm to room temperature thenstirred overnight.

The resulting precipitate was filtered off, then washed with 200 mL oftetrahydrofuran. After concentrating the filtrates to about 150 mL undervacuum, 200 mL of ethyl acetate was added, followed by ice to cool thesolution. The cooled solution was washed with two 75 mL portions of 0.2Nhydrochloric acid, two 75 mL portions of 0.5N sodium hydroxide, oneportion of 75 mL of brine, then was dried over anhydrous magnesiumsulfate. Upon concentration in vacuum, 22.7 g (70% yield) of solidBOC-N^(g) -nitroarginine N-methyl-O-methylcarboxamide was recovered.Thin layer chromatographic analysis in 9:1 dichloromethane/methanol(silica gel) showed one spot.

A flask was placed under a nitrogen atmosphere and cooled to -50° C.,then charged with 70 mL (70 mmole) of 1N lithium hydride (intetrahydrofuran) and 500 mL of dry tetrahydrofuran. 50 mL of a solutioncontaining 66 mmole of BOC-N^(g) -nitroarginineN-methyl-O-methylcarboxamide in dry tetrahydrofuran was slowly addedwhile the temperature of the reaction mixture was maintained at -50° C.After allowing the reaction mixture to warm to 0° C. by removal of thecooling, it was recooled to -30° C., at which temperature, 100 mL (0.2mole) of 2N potassium bisulfate was added with stirring over about a 10to 15 minute period. The reaction mixture was then allowed to stir atroom temperature for 2 hours. After filtering off the precipitate, thefiltrate was concentrated to 100 mL under vacuum. The concentrate wascombined with 200 mL ethyl acetate, then washed with two 50 mL portionsof 1N hydrochloric acid, two 50 mL portions of saturated sodiumbicarbonate, one 50 mL portion of brine, then was dried over anhydrousmagnesium sulfate. The mixture was concentrated under vacuum to yield13.6 g (70%) of the titled compound.

EXAMPLE 2 Preparation of Trans-4-(Amino methyl)-cyclohexane carboxylicacid benzyl ester para-toluenesulfonate salt (2) ##STR11##

Trans-4-(aminomethyl)-cyclohexane carboxylic acid (50 g (0.318 moles)),p-toluenesulfonic acid (61.7 g (0.324 mmoles)), benzyl alcohol (250 mL;2.4 moles) and toluene (250 mL) were combined and stirred. The resultingmixture was refluxed for 24 hours and the liberated water was removedazeotropically using a Dean-Stark apparatus. A clear solution wasobtained after 5 hours of refluxing. The solution was allowed to cool toroom temperature and the product crystallized. The mixture was vacuumfiltered, washed with ether and dried in a vacuum oven to give 128.12 g(96% yield) of the above-identified product. ¹ H NMR (CD₃ OD) δ1.05 (,2H), 1.43 (m, 2H), 1.59 (m, 1H), 1.85 (m, 2H), 2.03 (m, 2H), 2.33 (m,1H), 2.35 (s, 3H), 2.75 (d, 2H, 5.09 (s, 2H), 7.23 (d, 2H), 7.32 (m,5H), 7.69 (d, 2H). M.P. 154°-156° C.

Reference: Greenstein, Jesse P.; Winitz, Milton, Chemistry of the AminoAcids., vol. 2, p. 942, (1986).

EXAMPLE 3 Preparation of 1-t-Butoxycarbonylsemicarbozidyl-trans-4-methylcyclohexane carboxylic acid benzyl ester (4) ##STR12##

Carbonyldiimidazole (CDI) (3.24 g (0.02 moles) was dissolved in 45 mL ofdimethylformamide (DMF) at room temperature under nitrogen. To thatmixture, a solution of 2.48 g (0.02 moles ) t-butyl carbazate in 45 mLDMF was added dropwise. Next 8.38 g (0.02 moles ) of solid benzyl ester2 (the product of Example 2) was added, followed by the dropwiseaddition of 3.06 mL of triethylamine (TEA) over a 30 minute period. Thereaction mixture was allowed to stir at room temperature under nitrogenfor one hour. Water (100 mL) was added to the mixture and this mixturewas extracted three times with 50 mL of ethyl acetate. The ethyl acetatelayers were combined and extracted two times each with 75 mL 1N HCl, H₂O, 5% NaHCO₃, and saturated NaCl, and then dried with MgSO₄. The mixturewas filtered and the solution was concentrated to give an oil. This oilcrystallized on standing. This material may be purified further byrecrystallization, but may be used directly in the next proceduredescribed in Example 4 without additional purification and/or isolation.M.P.=106°-108° C. (EtOAc/hexane) ¹ H NMR (CDCl₃) δ0.94 (m, 2H), 1.42 (m,2H), 1.45 (s, 9H), 1.81 (m, 2H), 2.02 (m, 2H), 2.27 (m, 1H), 3.17 (t,2H), 5.09 (s, 2H), 5.51 (t, 1H), 6.46 (s, 2H), 7.34 (m, 4H).

EXAMPLE 4 Preparation of1-(t-Butoxycarbonyl)-semicarbazidyl-trans-4-methyl-cyclohexanecarboxylic acid (5) ##STR13##

To the crude BOC-benzyl ester 4 (product of Example 3), 250 mL ofmethanol (MeOH) and 500 mg of 10% palladium on activated carbon wereadded. After shaking on the hydrogenator for one hour at 5 psi, themixture was filtered with Celite through a fine fritted filter. Thesolution was concentrated to a foam, methylene chloride was added and aprecipitate formed. The mixture was kept 5° C. for 565 hours. Thecrystallized material was filtered with ether and 4.0 g of crude productwas obtained (12.7 mmoles; yield: 62% overall yield from compound 2.) ¹H NMR (CD₃ OD), δ0.96, (m, 2H), 1.42 (m, 2H), 1.46 (s, 9H), 1.82 (m,2H), 1.97 (m, 2H), 2.18 (m, 1H), 3.0 (t, 2H). M.P.=185°-189° C.

EXAMPLE 5 Preparation of Semicarbazidyl-trans-4-methyl cyclohexanecarboxylic acid trifluoroacetate salt ##STR14##

Compound 5 (the product of Example 4), (315 mg (1 mmole)) was added to10 mL of trifluoroacetic acid (TFA) at 0° C. and the resulting solutionwas allowed to stir for 30 minutes. After this time the solution wasadded dropwise to 75 mL of etcher. A precipitate formed. The mixture wasfiltered and washed with ether. Weight of crude product was 254 mg (0.77mmoles; yield (77%)). ¹ H NMR (CD₃ OD), δ1.0 (m, 2H), 1.38 (m, 2H), 1.43(m, 1H), 1.84 (m, 2H), 2.01 (m, 2H), 2.22 (m, 1H), 2.04 (d, 2H). M.P.=154°-156° C.

EXAMPLE 6 Preparation of alpha-N-(t-Butoxycarbonyl)-N^(g)-nitro-argininal-semicarbazonyl-trans-4-methyl-cyclohexane carboxylicacid (7) ##STR15##

A solution of 13.7 g (41.6 mmoles) of the product of Example 5, 18.0 g(˜59 mmoles) of crude 1 (the product of Example 1) in 135 mL ethanolcontaining 45 mL of water, was treated with 9.41 g (69 mmoles) of NaOAcand refluxed for one hour. This solution was allowed to cool and thenpoured into 0.1N HCl and extracted three times with ethyl acetate. Thecombined organic phase was washed with water, then brine, dried (overMgSO₄) and concentrated to a small volume. This cloudy mixture wasallowed to set overnight at 5° C. to precipitate the product, which wasisolated by filtration and dried under vacuum. This gave 9.9 g., 47%yield based on 6. ¹ H NMR (CD₃ OD) δ1.0 (m, 2H), 1.43 (s, 9H), 1.45-2.20(m, 13H), 3.09 (d, 2H), 3,30 (m, 2H), 4.18 (bs, 1H), 7.10 (d, 1H).M.P.=162°-163° C.

EXAMPLE 7 Synthesis of Semicarbazine Solid Support

A. Procedure A:

Coupling of Amino Acid Aldehydes to Modified Resin

(1) Place 0.8 g (0.5 mmoles, 0.62 g/ml) of Methylbenzhydrylamine (MBHA)resin in a reaction vessel. Note: All washes require 10 mL of solventwith agitation for 1.2 minutes. (2) Wash 1 time with dichloromethane(DCM). (3) Wash 3 times with dimethylformamide (DMF). (4) Wash 2 timeswith 10% diisopropylethylamine (DIEA)/DMF (5) Wash 4 times with DMF. (6)Add: 5 mL DMF

1 mmole 4-Methylmorpholine (NMM)=102 μl

1 mmole Benzotriazol-1-yloxy-tris-(dimethylamino)phosphonium-hexafluorophosphate (BOP reagent)=443 mg.

1 mmole compound 5 (FIG. 1) from Example 4=315 mg

(7) Mix on rotating wheel for 1 hour.

(8) Wash 3 times with DMF.

(9) Remove aliquot for ninhydrin test. Quantitate % coupling.

(10) Wash 3 times with DCM.

(11) Add 50% trifluoroacetic acid (TFA)/DCM and stir for 20 min.

(12) Wash 3 times with DCM. (Resin 6).

(13) Wash 2 times with 10% DIEA/DMF.

(14) In 5 mL DMF add 6 mmole=1.82 g BOC-nitro-argininal.

(15) Quantitatively transfer mixture to a test tube.

(16) Heat to 40° C. in an oil bath while bubbling in nitrogen gas forstirring.

(17) Let reaction proceed for 30 hours.

(18) Remove aliquot and wash 2 times with DCM. Note: Steps 19-23 pertainto the aliquot.

(19) Add 10 mL 50% TFA/DCM and stir for 20 min.

(20) Wash 3 times with DMF.

(21) Wash 2 times with 10% DIEA/DMF.

(22) Wash 3 times with DMF.

(23) Ninhydrin as for step 9: quantitate % coupling.

(24) Acetylate the remaining resin.

Wash 3 times with DMF

Add 15 mL DMF

0.47 mL acetic anhydride

0.7 mL triethylamine (TEA)

Stir for 30 minutes

Wash 3 times each with: DMF, DCM, MeOH and either.

(25) Let dry and weigh.

Synthesis of Semicarbazone Solid Support

B. Procedure B

(1) Place 0.8 g (0.5 mmoles, 0,62 g/mol) of Methylbenzhydrylamine (MBHA)resin in a reaction vessel. Note: all washes require 10 mL of solventwith agitation for 1-2 minutes.

(2) Wash 1 time with dichloromethane (DCM).

(3) Wash 3 times with dimethylformamide (DMF).

(4) Wash 2 times with 10% diisopropylethylamine (DIEA)/DMF

(5) Wash 4 times with DMF.

(6) Add: 5 mL DMF

1 mmole 4-Methylmorpholine(nmm)=102 μl

1 mmole Benzotriazol-1-yloxy-tris-(dimethylamino)phosphonium-hexafluorophosphate (BOP reagent)=443 mg.

1 mmole alpha-(4-butoxycarbonyl)-N^(g)-niroargininal-semicarbazonyl-trans-4-methyl-cyclohexane carboxylic acid7=500 mg

(7) Mix on rotating wheel on 16 hours.

(8) Wash 3 times with DMF.

(9) Wash 2 times with 10% DIEA/DMF.

(10) Wash three times with DMF.

(11) Wash successively with DCM, MeOH and ether.

(12) Let dry and weigh.

The resulting resin 8 obtained by Procedure B showed 98-99% couplingyield by ninhydrin.

Both procedures A and B will give the resin 8 shown in FIG. 1. Thisresin can then be extended at the N-terminus with amino acids or aminoacid analogs on a conventional peptide synthesizer using standard t-BOCmethodology. The resulting protected peptide analog can be cleaved fromsupport with formaldehyde, and deprotected with hydrogen/Pd, if desired.The nitro group can be removed from the guanidine group withoutreduction of the aldehyde.

EXAMPLES OF AUTOMATED SYNTHESIS OF PEPTIDE ALDEHYDES

The automated synthesis of peptide aldehydes was performed on an AppliedBiosystems model 430A peptide synthesizer using the BOC chemistryconditions in the 430A users manual. The following examples arerepresentative only. It will be apparent to those skilled in the artthat the use of other alpha-amino protecting groups, different cleavageor work-up conditions, or purification schemes, may also be utilized. Itwill also be recognized that the synthesis of other peptide aldehydeswill often require the use of other side-chain protecting groups, orcombination of protecting groups, that are also compatible with theinstant process. It will also be noted that due to the reactive natureof the aldehyde group it may not be possible to synthesize and isolateall possible sequences of peptide aldehydes.

EXAMPLE 8 Synthesis of alpha-t-Butoxycarbonyl-D-Leu-L-Pro-L-Argininal##STR16##

The above-identified compound was prepared using 500 mg SAAA support 8,α-BOC amino acids and standard solid phase peptide synthesis asdescribed above. The BOC protecting groups were removed using a solutionof 50% TFA/DCM. The resin (support) was neutralized with 10%diisopropylethylamine in DCM. Coupling of amino acids to support reagent(and growing amino-acid-support chain) was performed in DMF with DCC and1-hydroxybenztriazole. To 470 mg of the resulting peptide analog resin,5 mL tetrahydrofuran (THF), 1 mL acetic acid, 1 mL formaldehyde and 100μl 1N HCl are combined and stirred for about one hour. The solution isfiltered and washed with 10 mL THF. The filtrate is diluted with 100 mLwater and extracted with ethyl acetate. The ethyl acetate phase iswashed with brine, dried (magnesium sulfate), and concentrated. Thenitro protecting group and other hydrogen-removable protecting groupsare removed by hydrogenation in 10 mL 10% water/methanol with 300 μl 1NHCl and 200 mg palladium on carbon at 5 psi for 45 minutes. The mixtureis filtered through a fine fritted filter with Celite, washed withmethanol/water and concentrated to give the crude peptide aldehyde.

The resulting peptide aldehyde can then be further isolated by C-18reverse phase HPLC, using an aqueous/acetonitrile (0.01% TFA) system togive the corresponding TFA salts. The product is then purified usingreverse phase high performance chromatography on a 10 micron 300angstrom pore size C-18 packing. The column was eluted with an aqueousgradient of 0.1% trifluoroacetic and acetonitrile, gradient going from5% to 40% acetonitrile containing 0.01% trifluoroacetic acid.Lyophilization of the appropriate fractions gave the above-identifiedpeptide aldehyde as the trifluoroacetate salt. FAB massspectrum:calculated MW=468, observed MW=468.

EXAMPLE 9 Synthesis of alpha-t-Butoxycarbonyl-D-Phe-L-Pro-L-Argininal##STR17##

The above-identified compound was prepared using SAAA support 8, α-BOCamino acids and standard solid phase peptide synthesis as described inExample 8. The resin was cleaved, and deprotected, as described inExample 8. The crude product was purified using reverse phase highperformance chromatography as described in Example 8. FAB mass spectrum:calc. mw=502; obs. mw=502.

EXAMPLE 10 Synthesis of alpha-t-Butoxycarbonyl-L-Leu-L-Pro-L-Argininal##STR18##

The above-identified compound was prepared using SAAA support 8, α-BOCamino acids and standard solid phase peptide synthesis as described inExample 8. The resin was cleaved, and deprotected, as described inExample 8. The crude product was purified using reverse phase highperformance chromatography as described in Example 8. FAB mass spectrum:calc. mw=468; obs. mw=468.

EXAMPLE 11 Synthesis of alpha-t-Butoxycarbonyl-L-Leu-L-Tyr-L-Argininal##STR19##

The above-identified compound was prepared using SAAA support 8, α-BOCamino acids, 2-bromobenzyloxy-carbonyl protected tyrosine, and standardsolid phase peptide synthesis as described in Example 8. The resin wascleaved, and deprotected, as described in Example 8. The crude productwas purified using reverse phase high performance chromatography asdescribed in Example 8. FAB mass spectrum: calc. mw=534; obs. mw=534.

EXAMPLE 12 Synthesis of alpha-t-Butoxycarbonyl-L-Leu-L-Phe-L-Argininal##STR20##

The above-identified compound was prepared using SAAA support 8, α-BOCamino acids and standard solid phase peptide synthesis as described inExample 8. The resin was cleaved, and deprotected, as described inExample 8. The crude product was purified using reverse phase highperformance chromatography as described in Example 8. FAB mass spectrum:calc. mw=518; obs. mw=518.

EXAMPLE 13 Synthesis of alpha-t-Butoxycarbonyl-L-Asp-L-Pro-L-Argininal.##STR21##

The above-identified compound was prepared using SAAA support 8, usingα-BOC amino acids, benzyl ester protection of aspartic acid and standardsolid phase peptide synthesis as described in Example 8. The resin wascleaved, and deprotected, as described in Example 8. The crude productwas purified using reverse phase high performance chromatography asdescribed in Example 8. FAB mass spectrum: calc. mw=470; obs. mw=470.

EXAMPLE 14 Synthesis of alpha-t-Butoxycarbonyl-D-Leu-L-Ser-L-Argininal##STR22##

The above-identified compound was prepared using SAAA support 8, usingα-BOC amino acids, O-benzyl protection for serine, and standard solidphase peptide synthesis as described above. The resin was cleaved, anddeprotected, as described below. The crude product was purified usingreverse phase high performance chromatography as described in Example 8.FAB mass spectrum: calc. mw=458; obs. mw=458.

EXAMPLE 15 Synthesis of alpha-t-Butoxycarbonyl-L-Phe-L-Trp-L-Argininal##STR23##

The above identified compound was prepared using SAAA support 8, usingα-BOC amino acids, N-formyl-indole-Trp and standard solid phase peptidesynthesis as described in Example 8. The resin was treated withpiperidine using standard conditions, cleaved, and deprotected, asdescribed in Example 8. The crude product was purified using reversephase high performance chromatography as described in Example 8. FABmass spectrum: calc. mw=591; obs. mw=591.

EXAMPLE 16 Synthesis of alpha-t-Butoxycarbonyl-L-Leu-D-Pro-L-Argininal##STR24##

The title compound was prepared using SAAA support 8, α-Boc amino acidsand standard solid phase peptide synthesis as described in Example 8.The resin was cleaved, and deprotected, as described in Example 8. Thecrude product was purified using reverse phase high performancechromatography as described for Example 8. FAB mass spectrum: calc.mw=468; obs. mw=468.

EXAMPLE 17 Cleavage of Peptide Aldehyde from Resin

470 mg of peptide aldehyde coupled to support (as prepared according toExamples 8-16), 5 ml THF, 1 mL acetic acid, 1 mL aqueous formaldehyde,and 100 ul 1N HCl are combined and stirred for 1 hour. The solution isfiltered and washed with 10 mL THF and the filtrate is diluted with 100mL of water and extracted with ethyl acetate. The ethyl acetate phase iswashed with brine, dried (MgSO₄) and concentrated. The nitro group andother hydrogen removable protecting groups are removed, when it isdesirable, by hydrogenation in 10 mL 10% H₂ O/MeOH with 300 μl 1N HCland 200 mg activated palladium on carbon at 5 psi for 45 minutes. Themixture is filtered through a fine fritted filter with Celite, washedwith MeOH/water and concentrated to give the crude peptide. Theresulting peptide aldehyde can then be purified by C-18 reverse phaseHPLC, using an aqueous/acetonitrile (0.01% TFA) system to give thecorresponding TFA salts.

EXAMPLE 18

Preparation of α-N-t-butoxycarbonyl-L-leucinal ##STR25## A. Preparationof α-t-butoxycarbonyl-L-leucine-N-methyl-O-methylcarboxamide.

A 1-L, three-necked, round-bottom flask is equipped with a mechanicalstirrer, an electronic digital thermometer, and a graduated additionalfunnel. The flask is charged with 39.1 g (0.4 moles) ofN,O-dimethylhydroxylamine hydrochloride (Aldrich Chemical Co.) and 236mL methylene chloride. The suspension is stirred and cooled to 2° C.with an ice-water bath. N-methylpiperidine (Aldrich Chemical Co.), 48.8mL (0.41 moles), is placed in the addition funnel and added dropwisewhile the temperature is maintained at about 2° C. (±2°). A clear,colorless solution results which is kept cold and is used in thefollowing reaction.

A 5-L, three-necked, round-bottomed flask is equipped with a mechanicalstirrer, thermometer, and an addition funnel with drying tube, the flaskis charged with 100 g (0.4 moles) of α-t-butoxylcarbonyl-L-leucinehydrate (Bachem, Inc.), 458 mL tetrahydrofuran (Fisher Scientific Co.)and 1.8 L methylene chloride. A clear solution results on stirring,which is cooled to -20° C. (±2°) by immersing the flask in a dryice-2-propanol bath. N-methyl-piperidine, 48.8 mL (0.41 moles), isplaced in the addition funnel and is added rapidly to the mixture, whilethe temperature is allowed to rise to about -12° C. (±2°). Methylchloroformate (Aldrich Chemical Co.), 31 mL (0.4 moles) is then placedin the addition funnel and added rapidly to the mixture with goodstirring, while the temperature is maintained at about -12° C. (±2°).Two minutes later the above-described solution ofN,O-dimethylhydroxylamine is added. The cooling bath is removed and theclear solution is allowed to warm to room temperature over about 4 hours(and may be stirred overnight for convenience). The solution is thencooled to about 5° C. and extracted with two 500 mL portions of aqueous0.2N hydrochloric acid and two 500 mL portions of aqueous 0.5N sodiumhydroxide, while maintaining the organic phase at about 5° C. to about15° C. during the extractions. The solution is washed with 500 mL ofsaturated aqueous sodium chloride solution, dried over magnesium sulfateand concentrated on a rotary evaporator at a bath temperature of about30°-35° C. The residue is further evacuated on a Varian 5500 instrument(using a 250 mm×4.6 mm I.D. Alltech C-18 column with 60:40 methanol:0.5MNH₄ H₂ PO₄ as the mobile phase, UV detection at 210 nm).

B. Preparation of α-N-tert-butoxycarbonyl-L-leucinal

A 5-L, four-necked, round-bottomed flask is equipped with a mechanicalstirrer, a thermometer, a pressure-equalizing addition funnel, and anair-cooled condenser fitted with an argon blanket adapter. The flask ischarged under an argon blanket with 17.7 g (95% pure, 0.44 moles)lithium aluminum hydride (Alfa Products, Morton/Thiokol Inc.) and 1.5 Lanhydrous ethyl ether (Fisher Scientific Co.). The resulting suspensionis stirred at room temperature for about one hour or until most of thesolid is finely dispersed. The flask is immersed in a dry ice-2-propanolbath and the suspension is cooled to about -45° C. A solution of theα-t-butoxycarbonyl-L-leucine N-methyl-O-methylcarboxamide (preparedaccording to paragraph A above), in 300 mL anhydrous ethyl ether isplaced in the addition funnel and added to the lithium aluminum hydridesuspension (which is cooled to about -45° C. before the addition) in asteady stream while maintaining the reaction temperature at about -35°C. (±3°). After the addition is complete, the cooling bath is removedand the mixture is stirred and is allowed to warm to about ±5°. Then,the mixture is once again cooled to about -35° C. and a solution of 96.4g (0.171 moles) of sodium bisulfate (Matheson, Coleman and Bell, asaturated aqueous solution is obtained after stirring overnight) in 265mL deionized water is placed in the addition funnel. The sodiumbisulfate solution is added cautiously at first and then rapidly, whilethe temperature is allowed to rise to about -2° C. (±3°). The coolingbath is removed and the mixture is stirred for about one hour. Thereaction mixture is filtered through a 2 inch pad of celite. The filtercake is washed with two 500 mL portions of ethyl ether. The combinedether layers are washed in sequence with three 350 mL portions of cold(about 5° C.) 1N hydrochloric acid, two 350 mL portions of saturatedaqueous sodium bicarbonate solution, and 350 mL saturated sodiumchloride solution. The organic solution is dried over magnesium sulfateand evaporated on a rotary evaporator (bath at 30° C.), to give aresidual, slightly cloudy syrup. The product is stored in a freezer(about -17° C.) prior to use, since it may racemize if stored at roomtemperature.

Reference:

Smart, B. E. (ed.), Org. Syn., Volume 67, pages 69-75 (1988).

EXAMPLE 19 Preparationalpha-N-(t-Butoxycarbonyl)-leucinal-semicarbazonyl-trans-4-methyl-cyclohexanecarboxylic acid ##STR26##

A solution of 13.7 g (41.6 mmoles) of the product of Example 5 and 8.81g (45 mmoles) of alpha-N-t-butoxycarbonyl-leucinal in 135 mL ethanolcontaining 45 mL of water, is treated with 9.41 g (69 mmoles) of sodiumacetate and refluxed for one hour. This solution is allowed to cool andthen is poured into 0.1N HCl and is extracted three times with ethylacetate. The combined organic phase is washed with water, then brine,dried (MgSO₄) and concentrated to a small volume. The product ispurified by crystallization or column chromatography on silica gel.

EXAMPLE 20 Preparation of Leucinal Semicarbazone Solid Support ##STR27##

The procedure of Example 7B is followed except that an equamolar amountofα-N-(t-butoxycarbonyl)-leucinal-semicarbazonyl-trans-4-methyl-cyclohexane-carboxylicacid (the product of Example 19) is used in place of the product ofExample 5. This procedure gives a solid support suitable for thesynthesis of peptide C-terminal leucinals.

EXAMPLE 21 Preparation ofalpha-N-(t-Butoxycarbonyl)-alaninal-semicarbazonyl-trans-4-methyl-cyclohexaneCarboxylic Acid ##STR28##

A solution of 1.04 g (6.0 mmoles) of the product of Example 5 and 2.90 g(8.8 mmoles) of alpha-t-butoxycarbonyl-alaninal in 36 mL of 15% aqueousethanol, was treated with 1.63 g (12.0 mmoles) of sodium acetatetrihydrate and the resulting solution was refluxed for one hour. Thissolution was allowed to cool and then poured into 0.1N HCl and extractedthree times with ethyl acetate. The combined organic phase was washedwith water, then brine, dried (MgSO₄) and concentrated to a smallvolume. The product was purified by crystallization from ethylacetate/hexanes. This gave 1.43 g (64% yield) of a white solid:mp=194°-196° C. ¹ H NMR (CDCl₃) δ1.01 (m, 2H), 1.31 (d, J=5 Hz, 3H),1.46 (s, 9H), 1.8-2.3 (m, 5H), 3.16 (bs, 2H), 4.38 (bs, 1H), 7.06 (d,J=3.5 Hz, 1H). Analysis; Calculated for C₁₇ H₃₄ N₄ O₅ : C, 55.1; H, 8.2;N, 15.1. Found: C, 53.7; H, 7.8; N, 14.0.

EXAMPLE 22 Preparation of Alaninal Semicarbazone Solid Support ##STR29##

In preparing the above-identified product, the procedure described inExample 7B is followed except that an equimolar amount of the product ofExample 21 is used in place of compound 7 (the product of Example 6).This procedure gives a solid support product suitable for the synthesisof peptide C-terminal alaninals, in 98-99.5% coupling yield (byninhydrin).

EXAMPLE 23 Preparation ofalpha-N-(t-Butoxycarbonyl)-valinal-semicarbazonyl-trans-4-methyl-cyclohexaneCarboxylic Acid ##STR30##

A solution of 2.3 g (10.5 mmoles) of the product of Example 5 and 5.5 g(16.7 mmoles) of alpha-N-t-butoxycarbonyl-valinal in 68 mL of 15%aqueous ethanol, was treated with 3.08 g (22.6 mmoles) of sodium acetatetrihydrate and the resulting solution was refluxed for one hour. Thissolution was allowed to cool and then poured into 0.1N HCl and extractedthree times with ethyl acetate. The combined organic phase was washedwith water, then brine, dried (MgSO₄) and concentrated to a smallvolume. The product was purified by crystallization from ethylacetate/hexanes. This gave 3.83 g (91% yield) of a white solid:mp=144°-145° C. ¹ H NMR (CD₃ OD) δ0.95 (d, J=7 Hz, 6H), 1.01 (m, 2H),1.46 (s, 9H), 1.8-2.30 (m, 5H), 3.10 (bs, 2H), 3.98 (bs, 1H), 7.12 (d,J=3.5 Hz, 1H). Analysis; Calculated for C₁₉ H₃₄ N₄ O₅ : C, 57.3; H, 8.6;N, 14.1. Found: C, 56.3; H, 8.3; N, 13.7.

EXAMPLE 24 Preparation of Valinal Semicarbazone Solid Support ##STR31##

In preparing the above-identified product, the procedure described inExample 7B is followed except that an equimolar amount of the product ofExample 23 is used in place of compound 7 (the product of Example 6).This procedure gives a solid support product suitable for the synthesisof peptide C-terminal valinals, in 98-99.5% coupling yield (byninhydrin).

EXAMPLE 25 Preparation ofalpha-N-(t-Butoxycarbonyl)-phenylalaninal-semicarbazonyl-trans-4-methyl-cyclohexaneCarboxylic Acid ##STR32##

A solution of 2.5 g (10.0 mmoles) of the product of Example 5 and 3.6 g(11.0 mmoles) of alpha-N-t-butoxycarbonyl-phenylalaninal in 60 mL of 15%aqueous ethanol, was treated with 2.72 g (20.0 mmoles) of sodium acetatetrihydrate and the resulting solution was refluxed for one hour. Thissolution was allowed to cool and then poured into 0.1N HCl and extractedthree times with ethyl acetate. The combined organic phase was washedwith water, then brine, dried (MgSO₄) and concentrated to a smallvolume. The product was purified by crystallization from ethylacetate/hexanes. This gave 3.4 g (78% yield) of a white solid:mp=173°-174° C. ¹ H NMR (CD₃ OD) δ1.05 (m, 2H), 1.41 (s, 9H), 1.8-2.30(m, 5H), 2.9-3.1 (m, 4H), 4.43 (bs, 1H), 7.1-7.3 (d, 6H). Analysis;Calculated for C₂₃ H₃₄ N₄ O₅ : C, 61.9; H, 7.7; N, 12.5. Found: C, 61.6;H, 7.7; N, 12.2.

EXAMPLE 26 Preparation of Phenylalaninal Semicarbazone Solid Support##STR33##

In preparing the above-identified product, the procedure described inExample 7B is followed except that an equimolar amount of the product ofExample 25 is used in place of compound 7 (the product of Example 6).This procedure gives a solid support product suitable for the synthesisof peptide C-terminal phenylalaninals in 98-99.5% coupling yield (byninhydrin).

EXAMPLE 27 Preparation of alpha-t-Butoxycarbonyl-L-Asp-(O-benzylester)-L-Pro-L-Valinal ##STR34##

The title compound was prepared using 500 mg valinal SAAA support(product of Example 24), α-BOC-L-proline, α-BOC-O-benzyl aspartic acidand standard solid phase peptide synthesis as described above. Asolution of 50% TFA/DCM was used to remove the BOC groups. The resin wasneutralized with 10% diisopropylethylamine in DCM. Coupling wasperformed in DMF with DCC and 1-hydroxybenztriazole. To 470 mg ofresulting peptide aldehyde resin (from above), 5 mL THF, 1 mL aceticacid, 1 mL formaldehyde, and 100 μl 1N HCl are combined and stirred for1 hour. The solution is filtered and washed with 10 mL THF and thefiltrate is diluted with 100 mL of water and extracted with ethylacetate. The ethyl acetate phase is washed with brine, dried (MgSO₄) andconcentrated. The resulting peptide aldehyde can then be purified byC-18 reverse phase HPLC, using an aqueous/acetonitrile (0.1% TFA) systemto give the corresponding TFA salts. The crude product was purifiedusing reverse phase high performance chromatography on a 10 micron 300angstrom pore size C-18 packing. The column was eluted with a aqueousgradient of 0.01% trifluoroacetic acid gradient going from 5% to 60%acetonitrile containing 0.01% trifluoroacetic acid. Lyophilization ofthe appropriate fractions gave the title compound as itstrifluoroacetate salt. FAB mass spectrum: calculated mw=503; obs.mw=503.

What is claimed is:
 1. A compound of the formula: ##STR35## wherein (a)A is a non-reactive hydrocarbyl group having from 2 to about 15 carbonatoms; and(b) Z is selected from ##STR36## wherein Pr is a protectinggroup removable under non-adverse conditions and R₁ is hydrogen; oralkyl, cycloalkyl, aryl or aralkyl, optionally substituted with 1 to 3substituents independently selected from hydroxy, alkoxy, sulfhydryl,alkylthio, carboxy, amide, amino, alkylamino, indolyl,3-N-formylindolyl, benzyloxy, halobenzyloxy, guanido, nitro-guanido oroptionally substituted imidazolyl substituted with alkoxyalkyl; and alkis an alkylene group of about 3 to about 12 carbon atoms optionallysubstituted with 1 to 3 substituents independently selected fromhydroxy, alkyl, aryl or guanido; and provided that any functional groupsof R₁ or alk which are reactive under conditions of peptide synthesisare optionally protected by a protecting group which is removable undernon-adverse conditions.
 2. A compound according to claim 1 wherein A hasfrom about 5 to about 10 carbon atoms.
 3. A compound according to claim2 wherein Z is --NH--Pr.
 4. A compound according to claim 3 wherein A isa divalent radical selected from cycloalkylene, arylene and aralkylene,all having from about 5 to about 8 carbon atoms and optionallysubstituted with from 1 to 5 alkyl groups.
 5. A compound according toclaim 2 wherein Z is: ##STR37##
 6. A compound according to claim 5wherein A is a divalent radical selected from cycloalkylene, arylene andaralkylene all having from about 5 to about 8 carbon atoms andoptionally substituted from 1 to 5 alkyl groups.
 7. A compound accordingto claim 6 wherein A is 1,4-cyclohexylene, 1,3-cyclohexylene,1,3-phenylene, or 1,4-phenylene, 1,3-xylylene or 1,4-xylylene.
 8. Acompound according to claim 7 wherein R₁ is hydrogen, alkyl of 1 to 12carbon atoms, cycloalkyl of about 5 to 8 carbon atoms or aryl or aralkylof about 5 to 10 carbon atoms, all optionally substituted with asubstituent independently selected from hydroxy, alkoxy, carboxy, amino,amidophenyl, phenoxy, guanido, nitroguanido, imidazolyl or indolyl.
 9. Acompound according to claim 7 wherein R₁ is selected from the sidechains of glycine, alanine, valine, leucine, isoleucine, serine,threonine, cysteine, methionine, glutamic acid, aspartic acid,glutamine, asparagine, lysine, arginine, histine, phenylalanine,tyrosine and tryptophan.
 10. A compound according to claim 9 wherein Ais trans-4-methyl-1,4-cyclohexylene, and R₁ is the side chain of N^(g)-nitroarginine.
 11. A compound according to claim 2 wherein Z is:##STR38##
 12. A compound according to claim 11 wherein A is a divalentradical selected from cycloalkylene, arylene and aralkylene, all havingfrom about 5 to 8 carbon atoms and optionally substituted with from 1 to5 alkyl groups.
 13. A compound according to claim 12 wherein alk ispropylene or hydroxy-propylene.
 14. A compound according to claim 13wherein A is 1,3-cyclohexylene; 1,4-cyclohexylene; 1,3-phenylene or1,4-phenylene, 1,3-xylylene or 1,4-xylylene.